An understanding of transcriptional regulation is fundamental to elucidation of cellular controls during normal growth and during transformation. A unique feature of the E2F transcription factor is the regulation of activity by both viral and cellular signals. E2F activity is markedly increased in response to adenovirus infection. In addition, E2F is involved in the transcriptional response to cellular differentiation and proliferative signals, and E2F sites are present in several oncogenes and in other genes critical for cell growth. Understanding the molecular basis of E2F function could provide new insights into both cellular and viral transcriptional regulation. The adenovirus regulation of E2F provides an intriguing system in which the activity of a single transcription factor can be regulated by two distinct mechanisms. E2F is regulated by the adenovirus E1A and E4 proteins and utilizes both post-translational modification and protein-protein interactions, respectively. The experiments in this proposal are designed to probe the function and adenovirus regulation of E2F on the molecular and biochemical level. A first objective is a genetic dissection of the protein domains required for E2F function and regulation. A chief advantage of E2F is the availability of several well-characterized in vitro assays to rapidly score DNA binding, transcriptional activation, protein-protein interactions, and phosphorylation. A second objective is an assessment of the role of phosphorylation in modulating E2F activity in vivo. It has been demonstrated that phosphorylation is required for E2F activation in vitro, but the functional role in vivo is unknown. In vivo phosphorylation will be determined by immunoprecipitation of E2F from both 32P-labelled HeLa cells and then subsequent analysis be compared in nuclear extracts from both uninfected and infected HeLa cells. In conjunction with mutational analysis, the functional significance of phosphorylation sites can be assessed both in vivo and in vitro.